BackgroundCOPD is associated with cellular senescence and fibrosis. Extracellular vesicles (EVs) are membrane-derived vesicles involved in intercellular communication. EVs contain miRNAs, mRNA and proteins and have been implicated in COPD to induce senescence and the transition of fibroblast to myofibroblasts. This study examined whether EVs derived from COPD fibroblasts drive senescence in healthy recipient fibroblasts. Changes in expression of p21CIP1 and alpha-smooth muscle actin (αSMA) were chosen as markers of senescence and transition of fibroblasts to myofibroblasts respectively.MethodsLarge EVs, and small EVs were isolated from media from non-smoker (NS) and COPD fibroblasts cultured with or without H2O2. EVs were labelled with phk67 and uptake measured by flow cytometry. Healthy recipient fibroblasts were cultured with EVs or EV-free media for 24h and 48h and protein expression of p21CIP1 and αSMA measured using western blots and CXCL8 release by ELISA.ResultsThere was a time-dependent uptake of EVs into recipient cells with no difference between EVs from control or COPD fibroblasts with 91.8 ± 3.8% of recipient cells phk67 positive by 48h (n=4). Incubation of recipient fibroblasts (n=2–5) with large EVs from either non-smokers or COPD subjects did not alter the expression of p21CIP1 or αSMA at 24h. Similarly large EVs from fibroblasts exposed to H2O2 had no effect on these markers in recipient cells. By contrast, at 48h (figure 1), small EVs from COPD cells showed a trend to increased expression of p21CIP1 and EVs from both non-smokers and COPD subjects increased expression of αSMA. Incubation of recipient cells with large EVs from non-smoker fibroblasts that had been cultured with or without H2O2 increased release of CXCL8 (0.36±0.15ng/ml to 5.43±3.92ng/ml and 5.44±5.23ng/ml respectively) and small EVs from COPD fibroblasts induced CXCL8 release at 48h (0.36±0.15ng/ml to 3.75±3.16ng/ml).ConclusionsLarge and small EVs tend to increase the expression of p21CIP1 and αSMA in recipient fibroblasts. These results are confirmed by the uptake analysis showing that maximum uptake of EVs from both NS and COPD fibroblasts is reached after 48h. Altogether, these data suggest that EVs participate in COPD pathophysiology by spreading senescence in recipient fibroblasts.Abstract P208 Figure 1Effect of Large and Small EVs on p21CIP1 and αSMA Expression in NS Fibroblasts Stimulated for 48h. Healthy fibroblasts from non-smoker (NS) subjects were incubated with large and small EVs derived from healthy NS or COPD fibroblasts, derived from cells that had been cultured in the absence or presence of 100µM H2O2. Cells were also stimulated with media only (NT) and media containing H2O2 as controls. Cells were lysed after 48h (a, d) and the expression of p21CIP1 (b, e) and αSMA (c, f) was measured relative to β-actin expression and data presented as mean±SEM. Representative blots are presented in panels a and d.